1. Field of the Invention
The present invention relates generally to the field of molecular biology, and more specifically, to a method for inhibiting conidial germination and mycelial growth of fungi that live symbiotically with various species of bark beetles.
2. Description of the Related Art
The present invention addresses the need to control infestations of coniferous trees by different species of bark beetles. Bark beetles, such as the mountain pine beetle Dendroctonus ponderosae and the western pine beetle Dendroctonus brevicomis, are responsible for killing large numbers of coniferous trees over vast areas of western North America. Indeed, beetle outbreaks are the leading cause of pine tree mortality in North America, and since the outbreak began in the late 1990s, approximately 45 million acres of pine trees have been killed in United States alone.
Bark beetles colonize pine trees as a natural part of their life cycle. During mid-summer, so-called “pioneer” beetles initiate tree colonization by boring through the bark and releasing pheromones that attract additional male and female beetles to a tree. The congregating beetles then mate, excavate an egg chamber within the phloem layer, lay eggs and die. After hatching, the larval progeny develop within the tree (carving out a system of tunnels as they feed on the sapwood) and emerge the following summer as adults. Many bark beetles colonize weak or dying trees; however, in the current outbreak, mountain pine beetles are colonizing and killing healthy trees.
Most species of bark beetles are host to a variety of fungal species from the genera Grosmannia, Ophiostoma, Ceratocystiopsis and Ceratocystis (1). Many of these fungal species have an obligate symbiotic relationship with bark beetles; that is, the fungi are required for the survival of the host bark beetle, most likely by providing access to tree nutrients (2,3,4). The beetles carry their symbiotic fungi as conidia in mycangia, specialized structures located on their exoskeleton. During colonization of a pine tree, the bark beetles passively introduce fungal conidia to the tree interior. Through conidial germination and mycelial growth, the fungi are able to invade phloem layer and sometimes also the xylem of the tree, discoloring the wood and disrupting water flow in the tree. It is not clear whether the fungi have a direct causal role in tree death (through mycelial invasion of the wood, for example) or whether they contribute indirectly to tree death through their symbiotic relationship with the beetles. In any case, the chemical inhibition of fungal growth—that is, the inhibition of conidial germination and/or mycelial growth—may provide a novel means of managing, controlling or limiting bark beetle infestations of pine trees.
The present invention is based on the unexpected discovery that the small molecule 5-(p-Bromobenzylidine)-α-isopropyl-4-oxo-2-thioxo-3-thiozolidineacetic acid (hereafter referred to as “BH3I-1”) and four structurally related molecules, 2-[5-(4-chlorobenzylidene)-4-oxo-2-thioxo-1,3-thiazolidin-3-yl]-3-methylbutanoic acid (hereafter referred to as “BH3I-1-12”), 2-[5-(4-hydroxybenzylidene)-4-oxo-2-thioxo-1,3-thiazolidin-3-yl]pentanoic acid (hereafter referred to as “BH3I-1-66”), [5-(4-isopropylbenzylidene)-4-oxo-2-thioxo-1,3-thiazolidin-3-yl]acetic acid (hereafter referred to as “BH3I-1-68”) and 2-[5-(2-chlorobenzylidene)-4-oxo-2-thioxo-1,3-thiazolidin-3-yl]pentanoic acid (hereafter referred to as “BH3I-1-69”), inhibit the conidial germination and mycelial growth of fungal species that have an obligate symbiotic relationship with the mountain pine beetle (Dendroctonus ponderosae) and the western pine beetle (Dendroctonus brevicomis). The fungal species inhibited by BH3I-1, BH3I-I-12, BH3I-1-66, BH3I-1-68 and BH3I-1-69 include Grosmannia clavigera (symbiont of mountain pine beetle), Ophiostoma montium (symbiont of mountain pine beetle) and Ceratocystiopsis brevicomi (symbiont of western pine beetle).
The inventors do not think the inhibitory activity of BH3I-1 against bark beetle fungal symbionts is an obvious discovery in light of prior art. The inventors previously found that BH3I-1 inhibits morphogcnesis (i.e., the yeast-to-filamentous growth transition) in Candida ulbicans (5). C. albicans and the bark beetle fungal symbionts share only a distant evolutionary history. Comparative genomic analyses have placed C. albicans and G. clavigera (the mountain pine beetle symbiont) in different taxonomic subphyla with a most recent common ancestor of approximately 350 million years ago (6,7). Because it has been shown that anti-fungal susceptibility can vary widely even among species of the same genus (8), there is no basis to expect that an inhibitory molecule will be active on such evolutionarily divergent fungi as C. albicans and the bark beetle fungal symbionts.